Abstract
Abstract Hen egg white lysozyme, fully inactivated by the affinity-labeling reagent, the 2',3'-epoxypropyl β-glycoside of di-(N-acetyl-d-glucosamine), contained 1 mole of the label per mole of protein. Total enzymatic digestion of the reduced and carboxymethylated affinity-labeled enzyme afforded, as a major radioactive product, a compound composed solely of aspartic acid and glucosamine at the molar ratio of 1:2. Peptic digestion afforded a single radioactive peptide corresponding to the sequence [see PDF for sequence] in the enzyme. Digestion of this peptide with Clostridium histolyticum aminopeptidase gave a radioactive pentapeptide the composition, partial structure, and other properties of which corresponded to the sequence [see PDF for sequence] in hen egg white lysozyme, and which contained 2 moles of glucosamine per mole of peptide. Our findings show that the affinity label is covalently bound to the enzyme via the β-carboxyl group of Asp 52 and are in accord with our conclusions from immunochemical (Maron, E., Eshdat, Y., and Sharon, N. (1972) Biochim. Biophys. Acta 278, 243) and x-ray crystallographic (Moult, J., Eshdat, Y., and Sharon, N. (1973) J. Mol. Biol. 75, 1) studies of the affinity-labeled hen egg white lysozyme.
Highlights
ACetyl-D-glUCOSamine), contained 1 mole of the label per mole of protein
Digestion of this peptide with Clostridium histolyticum aminopeptidase gave a radioactive pentapeptide the composition, partial structure, and other properties of which corresponded to the sequence G:i-Ser-Thr-Asp-Ty: in hen egg white lysozyme, and which contained 2 moles of glucosamine per mole of peptide
Our findings show that the affinity label is covalently bound to the enzyme via the fl-carboxyl group of Asp 52 and are in accord with our conclusions from immuno
Summary
Activities of hen egg white lysozyme and of the affinitylabeled enzyme were measured, essentially as described by Shugar [12], in a Beckman DB-G recording spectrophotometer equipped with a cell holder thermostated at 26”. ~o~o\~o.cH2.c~-;H. hydrolysate under reduced pressure in a desiccator, the amino acid composition was determined on an automated amino acid analyzer [17] with loads of 5 to 100 nmoles per amino acid. For the determination of glucosamine on the long column, the buffer change time was set at 75 min instead of 85 min. The glucosamine was analyzed on a medium-sized column.
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